beta-cell type-specific expression of the upstream glucokinase promoter was studied by transfection of fusion genes and analysis of DNA-protein interactions. A construct containing 1,000 bp of 5'-flanking DNA was efficiently expressed in HIT M2.2.2 cells, a beta-cell-derived line that makes both insulin and glucokinase, but not in NIH 3T3 cells, a heterologous cell line. In a series of 5' deletion mutations between bases -1000 and -100 (relative to a base previously designated +1), efficient expression in HIT cells was maintained until -280 bp, after which transcription decreased in a stepwise manner. The sequences between -180 and -1 bp contributing to transcriptional activity in HIT cells were identified by studying 28 block transversion mutants that spanned this region in 10-bp steps. Two mutations reduced transcription 10-fold or more, while six reduced transcription between 3- and 10-fold. Three mutationally sensitive regions of this promoter were found to bind to a factor that was expressed preferentially in pancreatic islet beta cells. The binding sites, designated upstream promoter elements (UPEs), shared a consensus sequence of CAT(T/C)A(C/G). Methylation of adenine and guanine residues within this sequence prevented binding of the beta-cell factor, as did mutations at positions 2, 3, and 5. Analysis of nuclear extracts from different cell lines identified UPE-binding activity in HIT M2.2.2 and beta-TC-3 cells but not in AtT-20, NIH 3T3, or HeLa cells; the possibility of a greatly reduced amount in alpha-TC-6 cells could not be excluded. UV laser cross-linking experiments supported the beta-cell type expression of this factor and showed it to be approximately 50 kDa in size. Gel mobility shift competition experiments showed that this beta-cell factor is the same that binds to similar elements, termed CT boxes, in the insulin promoter. Thus, a role for these elements (UPEs or CT boxes), and the beta-cell factor that binds to them, in determining the expression of genes in the beta cells of pancreatic islets is suggested.